BNLCL2 Cells Research Articles

MicroRNA-181a-5p alleviates acute liver failure in mice by inhibiting HMGB1.

MicroRNAs (miRNAs) control liver diseases, but the role of microRNA-181a-5p in acute liver failure (ALF) is unclear. In this study, the ALF model was generated by injection of D-galactosamine (D-GalN) and lipopolysaccharide (LPS). The levels of miRNAs were assessed by microarray and qRT-PCR. The expression of caspase 3 was detected as the marker of cell apoptosis in ALF by immunohistochemistry and western blot. The targeting of microRNA-181a-5p on the high mobility group box 1 (HMGB1) was verified by dual luciferase assay. The impact of microRNA-181a-5p and HMGB1 was explored by flow cytometry. Results showed that microRNA-181a-5p was significantly down-regulated by D-GalN/LPS in vivo and in vitro, while the level of HMGB1 was up-regulated after the challenge. Furthermore, microRNA-181a-5p overexpression attenuated cell apoptosis in D-GalN/TNF-treated BNLCL2 cells. MicroRNA-181a-5p could directly target HMGB1 mRNA and repress its expressions, in further HMGB1 is involved in microRNA-181a-5p effect on cell apoptosis of ALF. In conclusion, these findings demonstrate that microRNA-181a-5p regulates hepatocyte apoptosis via HMGB1 in the development of ALF, which may provide potential therapeutic targets for ALF. However, the precise underlying mechanism that connects microRNA-181a-5p and HMGB1 remains to be explored.

Role of the microRNA‑214/Bax axis in the progression of acute liver failure.

Acute liver failure (ALF) is a fatal liver disease characterized by severe hepatocyte destruction. MicroRNAs (miRNAs/miRs) have been reported to serve a key role in a number of liver diseases. Therefore, the aim of the present study was to investigate the role and underlying mechanism of miR-214 in ALF. ALF murine and hepatocyte models were established using D-galactosamine (D-GalN) and lipopolysaccharide (LPS) or D-GalN + tumor necrosis factor (TNF)-α, respectively. The expression levels of miR-214 and Bax were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and/or western blotting. Furthermore, an automatic biochemical analyzer was used to measure the levels of aspartate aminotransferase (AST) or alanine aminotransferase (ALT). The levels of TNF-α and interleukin (IL)-6 were detected by ELISA and RT-qPCR. In addition, TUNEL staining and flow cytometry were used to analyze cell apoptosis, and the protein expression of caspase-3 was determined by western blotting. It was identified that the levels of AST and ALT were increased and that hepatocyte apoptosis was enhanced in the D-GalN/LPS-stimulated group compared with the control. Furthermore, higher expression of caspase-3 was observed in the D-GalN/LPS-stimulated group. In addition, it was demonstrated that miR-214 was downregulated, while Bax was upregulated in D-GalN/LPS-stimulated mice and D-GalN/TNF-α-stimulated BNLCL2 cells. Moreover, in D-GalN/TNF-α-stimulated BNLCL2 cells, miR-214 overexpression suppressed apoptosis and decreased TNF-α and IL-6 levels, and these effects were reversed by the Bax plasmid. It was also identified that overexpression of miR-214 significantly decreased Bax mRNA and protein expression levels in vitro. Collectively, the present results suggested that miR-214 inhibited hepatocyte apoptosis during ALF development via targeting Bax, thus indicating that miR-214 may be a potential target for ALF treatment.

Metabolic effects of RUBCN/Rubicon deficiency in kidney proximal tubular epithelial cells

ABSTRACT Macroautophagy/autophagy is a lysosomal degradation system which plays a protective role against kidney injury. RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) inhibits the fusion of autophagosomes and lysosomes. However, its physiological role in kidney proximal tubular epithelial cells (PTECs) remains uncertain. In the current study, we analyzed the phenotype of newly generated PTEC-specific rubcn-deficient (KO) mice. Additionally, we investigated the role of RUBCN in lipid metabolism using isolated rubcn-deficient PTECs. Although KO mice exhibited sustained high autophagic flux in PTECs, they were not protected from acute ischemic kidney injury. Unexpectedly, KO mice exhibited hallmark features of metabolic syndrome accompanied by expanded lysosomes containing multi-layered phospholipids in PTECs. RUBCN deficiency in cultured PTECs promoted the mobilization of phospholipids from cellular membranes to lysosomes via enhanced autophagy. Treatment of KO PTECs with oleic acid accelerated fatty acids transfer to mitochondria. Furthermore, KO PTECs promoted massive triglyceride accumulation in hepatocytes (BNL-CL2 cells) co-cultured in transwell, suggesting accelerated fatty acids efflux from the PTECs contributes to the metabolic syndrome in KO mice. This study shows that sustained high autophagic flux by RUBCN deficiency in PTECs leads to metabolic syndrome concomitantly with an accelerated mobilization of phospholipids from cellular membranes to lysosomes. Abbreviations: ABC: ATP binding cassette; ACADM: acyl-CoA dehydrogenase medium chain; ACTB: actin, beta; ATG: autophagy related; AUC: area under the curve; Baf: bafilomycin A1; BAT: brown adipose tissue; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; BW: body weight; CAT: chloramphenicol acetyltransferase; CM: complete medium; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; CTRL: control; EGFP: enhanced green fluorescent protein; CTSD: cathepsin D; EAT: epididymal adipose tissue; EGFR: epidermal growth factor receptor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FA: fatty acid; FBS: fetal bovine serum; GTT: glucose tolerance test; HE: hematoxylin and eosin; HFD: high-fat diet; I/R: ischemia-reperfusion; ITT: insulin tolerance test; KAP: kidney androgen regulated protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor related protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MAT: mesenteric adipose tissue; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NDRG1: N-myc downstream regulated 1; NDUFB5: NADH:ubiquinone oxidoreductase subunit B5; NEFA: non-esterified fatty acid; OA: oleic acid; OCT: optimal cutting temperature; ORO: Oil Red O; PAS: Periodic-acid Schiff; PFA: paraformaldehyde; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PPARA: peroxisome proliferator activated receptor alpha; PPARGC1A: PPARG coactivator 1 alpha; PTEC: proximal tubular epithelial cell; RAB7A: RAB7A, member RAS oncogene family; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RT: reverse transcription; RUBCN: rubicon autophagy regulator; SAT: subcutaneous adipose tissue; SFC: supercritical fluid chromatography; SQSTM1: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; SV-40: simian virus-40; TFEB: transcription factor EB; TG: triglyceride; TS: tissue specific; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN: urea nitrogen; UQCRB: ubiquinol-cytochrome c reductase binding protein; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WAT: white adipose tissue.

Radical Scavenging Activities of Tannin Extracted from Amaranth (Amaranthus caudatus L.)

This study investigates the bioactivity of tannin from amaranth (Amaranthus caudatus L.) extracts. The antioxidant activities of the extracts from amaranth leaves, flowers, and seeds were evaluated. Tannin from leaves of amaranth has been evaluated for superoxide scavenging activity by using DPPH and ABTS(+) analysis, reducing power, protective effect against H2O2-induced oxidative damage in L-132 and BNL-CL2 cells, and inhibition of superoxide radical effects on HL-60 cells. At a concentration of 100 μg/ml, tannin showed protective effects and restored cell survival to 69.2% and 41.8% for L-132 and BNL-CL2 cells, respectively. Furthermore, at the same concentration, tannin inhibited 41% of the activity of the superoxide radical on HL-60 cells and 43.4% of the increase in nitric oxide levels in RAW 264.7 cells. The expression levels of the antioxidant-associated protein SOD-1 were significantly increased in a concentration-dependent manner in RAW 264.7 cells treated with tannin from amaranth leaves. These results suggest that tannin from the leaves of Amaranthus caudatus L. is a promising source of antioxidant component that can be used as a food preservative or nutraceutical.

Brazilein fromCaesalpinia sappanL. Antioxidant Inhibits Adipocyte Differentiation and Induces Apoptosis through Caspase-3 Activity and Anthelmintic Activities againstHymenolepis nanaandAnisakis simplex

Brazilein, a natural, biologically active compound from Caesalpinia sappan L., has been shown to exhibit anti-inflammatory and antioxidant properties and to inhibit the growth of several cancer cells. This study verifies the antioxidant and antitumor characteristics of brazilein in skin cancer cells and is the first time to elucidate the inhibition mechanism of adipocyte differentiation, cestocidal activities against Hymenolepis nana, and reduction of spontaneous movement in Anisakis simplex. Brazilein exhibits an antioxidant capacity as well as the ability to scavenge DPPH• and ABTS•+ free radicals and to inhibit lipid peroxidation. Brazilein inhibited intracellular lipid accumulation during adipocyte differentiation in 3T3-L1 cells and suppressed the induction of peroxisome proliferator-activated receptor γ (PPARγ), the master regulator of adipogenesis, suggesting that brazilein presents the antiobesity effects. The toxic effects of brazilein were evaluated in terms of cell viability, induction of apoptosis, and the activity of caspase-3 in BCC cells. The inhibition of the growth of skin cancer cells (A431, BCC, and SCC25) by brazilein is greater than that of human skin malignant melanoma (A375) cells, mouse leukemic monocyte macrophage (RAW 264.7 cells), and noncancerous cells (HaCaT and BNLCL2 cells). The anthelmintic activities of brazilein against Hymenolepis nana are better than those of Anisakis simplex.

Free Radical Scavenging Activity of 4-(3,4-Dihydroxybenzoyloxymethyl)phenyl-O-β-d-glucopyranoside from Origanum vulgare and Its Protection against Oxidative Damage

4-(3,4-Dihydroxybenzoyloxymethyl)phenyl-O-β-d-glucopyranoside (DBPG), a polyphenolic glycoside, isolated from Origanum vulgare has shown 1,1-diphenyl-2-picrylhydrazyl (DPPH(•))-scavenging capacity in previous work. This study demonstrated that DBPG exhibits antioxidant activity by a series of DPPH(•), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS(•+)), and superoxide anion radical (O(2)(•-)) radical-scavenging assays. The inhibition of lipid peroxidation (LP) by DBPG exceeded that by l-ascorbic acid (AA) in a liposome model system. Adding DBPG to mouse liver and brain tissue inhibited the formation of thiobarbituric acid reactive substances (TBARS) to a greater extent than did trolox. In the oxygen stress test, BNLCL2 and HaCaT cells pretreated with DBPG showed increased activities of glutathione peroxidase (GPx), perhaps as a result of reduction of the production of reactive oxygen species (ROS). These findings proved that DBPG had antioxidant activities and a cytoprotective effect in hepatocytes and keratinocytes, suggesting that DBPG may be a useful food and cosmetic additive.

Apigenin induces apoptosis via tumor necrosis factor receptor- and Bcl-2-mediated pathway and enhances susceptibility of head and neck squamous cell carcinoma to 5-fluorouracil and cisplatin

BackgroundApigenin, a natural plant flavone, may have chemopreventive and therapeutic potentials for anti-inflammatory, antioxidant, and anti-cancer. Nevertheless, the anti-tumor effect of apigenin on human head and neck squamous cell carcinoma (HNSCC) is not fully understood. MethodsThe antioxidant capacity and protective effects of apigenin against oxidative stress in murine normal embryonic liver BNLCL2 cells are examined. Cell viability, morphologic change, clonogenic survival, cell cycle distribution, reactive oxygen species (ROS) production, glutathione formation, and death receptors- and Bcl-2-mediated caspase pathways of HNSCC SCC25 cells and A431 cells with apigenin are investigated. ResultsApigenin inhibits the growth of SCC25 and A431 cells and induces cell cycle arrest in the G2/M phase. Apigenin has an antioxidant capacity as well as the ability to inhibit lipid peroxidation. It protects BNLCL2 cells against oxidative damage, and is potentially able to prevent cancer. Apigenin increases intracellular ROS levels and reduces levels of glutathione; it also induces cell apoptosis via tumor necrosis factor receptor (TNF-R)-, TNF-related apoptosis-inducing ligand receptor (TRAIL-R)-, and Bcl-2-mediated caspase-dependent cell death pathways in SCC25 cells. The combination of apigenin with 5-fluorouracil (5-Fu) or cisplatin induces the dramatic death of SCC25 cells. ConclusionsApigenin induces SCC25 cell apoptosis via the up-regulation of both TNF-R and TRAIL-R signaling pathways, and has a synergistic effect on the inhibition of cell proliferation in combination with 5-Fu or cisplatin. General significanceThese analytical findings suggest that apigenin may be a good therapeutic agent against HNSCC cells.

The regulatory role of microRNA-1187 in TNF-α-mediated hepatocyte apoptosis in acute liver failure

In the current study, we aimed at elucidating the regulatory mechanisms through which microR-1187 (miR-1187) participates in hepatocyte apoptosis in acute liver failure (ALF). An ALF model was induced with D-galactosamine (D-GalN) plus lipopolysaccharide (LPS) in BALB/c mice. The hepatic miRNA expression profile was detected by microarray analysis and verified by quantitative real-time PCR (qRT-PCR). The possible underlying mechanism was investigated in vitro using an embryonic murine hepatocyte cell line (BNLCL2) and miR-1187 mimic. Caspase-8 protein was detected by Western blotting and cell apoptosis was assayed by flow cytometry. Hepatic miR-1187 was down-regulated in ALF mice based on microarray data (P<0.001) and verified by qRT-PCR (P<0.01). Target scan revealed that caspase-8 was the putative target of miR-1187. In an in vitro study, miR-1187 showed the highest up-regulation in BNLCL2 cells transfected with the miR-1187 mimic at a 50 nM concentration for 12 h compared with cells transfected with the non-specific mimic (P<0.001). miR-1187 was down-regulated (P<0.01) but caspase-8 mRNA (P<0.01) as well as protein (P<0.05) were up-regulated in the BNLCL2 cells treated with D-GalN/TNF. Furthermore, overexpressed miR-1187 reduced caspase-8 expression at both the mRNA and protein levels significantly (P<0.01 and P<0.05 respectively), and significantly attenuated the apoptotic rate of BNLCL2 cells (P<0.05). We show that miR-1187 regulates hepatocyte apoptosis by targeting caspase-8. miR-1187 may serve as a potential therapeutic target for the treatment of ALF.

Novel phenolic glucoside, origanoside, protects against oxidative damage and modulates antioxidant enzyme activity

Our previous research demonstrated that a novel phenolic glucoside, origanoside, isolated from Origanum vulgare, exhibits significant antimelanogenesis activity. The present study was designed to investigate whether origanoside may serve as a quencher of oxidative insults and is involved in antimelanogenesis. It is found that origanoside exhibit the radical-scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS+) and superoxide anion radical (O2−), the reducing power, and the inhibition ability of lipid peroxidation (LP). Pretreatment with origanoside in hydrogen peroxide (H2O2)-treated BNLCL2 and HaCaT cells increased the activities of catalase (CAT) and glutathione peroxidase (GPx), potentially reducing the amount of reactive oxygen species (ROS) generation. These findings confirm the cytoprotective effect of origanoside in hepatocytes and keratinocytes, suggesting that origanoside exhibits antioxidant activities and may be a useful food additive.

Inhibition of Melanogenesis and Oxidation by Protocatechuic Acid from Origanum vulgare (Oregano)

Antioxidant and antimelanogenesis activities of protocatechuic acid (1) from Origanum vulgare (oregano) were investigated. The antioxidative capacity of 1 was confirmed from its free-radical-scavenging activities, inhibition of lipid peroxidation, and suppression of reactive oxygen species in H(2)O(2)-induced BNLCL2 cells. The inhibition by 1 of tyrosinase and DOPA oxidase activity and melanin production was possibly related to the down-regulation of melanocortin-1 receptor, microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related proteins-2, and tyrosinase-related proteins-1 expression in α-melanocyte-stimulating hormone-induced B16 cells. After a gel containing 1 was applied to mice, the values of L* slightly increased, and a* and erythema-melanin levels of skin were reduced by comparing the values of untreated control groups, indicating 1 can reduce melanin production. These results suggest that 1 may act as an effective quencher of oxidative attackers with antimelanogenesis properties.

Antioxidative characteristics and inhibition of α‐melanocyte‐stimulating hormone‐stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare

The antioxidant activities of vanillin and vanillic acid isolated from Origanum vulgare are investigated. These compounds may serve as agents for antimelanogenesis. Vanillic acid is a stronger antioxidant than vanillin, in terms of free radical scavenging activity, reducing power and inhibition of lipid peroxidation. The inhibition of cellular reactive oxygen species (ROS) in H(2)O(2)-treated BNLCL2 cells by vanillic acid exceeds that of ascorbic acid (AA) or trolox. In B16F0 cells stimulated with alpha-melanocyte-stimulating hormone (alpha-MSH), vanillic acid reduced cellular tyrosinase activity, DOPA oxidase and melanin contents, as well as down-regulated expressions of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related proteins 2 (TRP-2) and TRP-1. Vanillin did not express inhibition of tyrosinase activity. These results supported that vanillic acid is a significantly stronger antioxidant than vanillin and exhibited stronger antimelanogenesis performance because of the structural presence of the carboxyl group.

Antioxidant and antimelanogenic properties of rosmarinic acid methyl ester from Origanum vulgare

Rosmarinic acid methyl ester (Ov-8) was isolated form Origanum vulgare. The antioxidant activities of Ov-8 were evaluated by measuring radical-scavenging activities, reducing power, and inhibition of lipid peroxidation, using liposomes and mice tissue. The percentage inhibition, by Ov-8, of the activities of reactive oxygen species (ROSs) in H 2O 2-treated BNLCL2 cells was about 116.0–82.3% of that in H 2O 2-treated cells. The ability of Ov-8 to reduce tyrosinase, DOPA oxidase and melanin contents in B16 cells exceeded those of arbutin and l-ascorbic acid. Ov-8 reduced the expressions of the melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related proteins-2 (TRP-2) and TRP-1 in B16 cells. Applying Ov-8-gel to mice increased L ∗ and reduced the erythema ( E ∗) and melanin ( M ∗) indices in relation to those of the base-gel and untreated groups. These results suggest that Ov-8 exhibits antioxidant and depigmentation activities that may be useful in food additives and in the control of skin pigmentation.

Abstract 4086: Ultraconserved non-coding RNAs are involved in human hepatocellular cancer growth

Abstract Background: Non-coding RNAs (ncRNA) have recently been implicated in carcinogenesis. Highly conserved ncRNA expression across species suggests a biological function. A group of ncRNAs that are greater than 200 bp and highly conserved across species (ultraconserved regions) has been recently shown to be differentially expressed in human cancers but their role in hepatocarcinogenesis remains unclear. Our aim was to evaluate the expression and potential role of these ultraconserved RNA (ucRNA) in hepatocellular cancer (HCC). Methods: The global expression of 481 ucRNAs was analyzed in human non-malignant (HH) or malignant (HepG2) hepatocytes using a custom microarray. Expression of selected ucRNA was verified by real time PCR in HCC cell lines and by in situ hybridization in 30 human HCC tissues. Cells were transfected with siRNAs or plasmids over-expressing ucRNA to reduce or enhance their cellular expression. Anchorage dependent growth was evaluated using viable cell assay and anchorage independent growth was assessed by growth in soft agar using a fluorometric assay. Cell cycle progression was determined by flow cytometry. Results: 56 ucRNAs were aberrantly expressed with 33 increased and 23 decreased in HepG2 cells compared to HH (p&amp;lt;0.05). Of these, uc.338 was the most up-regulated (6.9-fold). uc.338 was significantly increased in several HCC cell lines (HepG2, Huh-7, PLC/PRF-5, SK-Hep-1, SNU-182, and SNU-449) compared to HH by real-time PCR. uc.338 expression was also detected in 73% of human HCC. Although uc.338 is located within the PCBP2 gene, the expression of PCBP2 mRNA did not correlate with that of uc.338 and inhibition of uc.338 did not reduce PCBP2 mRNA, suggesting independent regulation of these genes. Over-expression of uc.338 increased growth rate of HH compared to cells transfected with an empty vector. Huh-7 and HepG2 cells transfected with anti-uc.338 had slower growth rate compared to cells transfected with control-siRNA. Growth in soft agar was decreased by 40% in HepG2 cells transfected with anti-uc.338 compared to controls. Transfection with anti-uc.338 also increased the number of cells in sub-G1 phase and decreased the number of cells in S phase by 30 ± 1.3 % (p&amp;lt;0.001) compared to controls. To confirm the conservation of uc.338 in different species, we assessed its expression in murine BNL-CL2 cells (embryonic liver mouse cells) and BNL-SVA.8 cells (SV40-transformed BNL-CL2 cells with an increased growth potential). uc.338 was over-expressed by 2-fold in BNL-SVA.8 cells compared to BNL-CL2 cells (p:0.001). Inhibition of uc.338 by siRNA reduced anchorage-dependent growth by 65% at 72 hours in BNL-SVA.8 cells (p: 0.0001). Conclusion: These studies identify a novel non-coding RNA uc.338 that is increased in expression and modulates cell growth in HCC in a species-independent fashion. These studies indicate a functional role for ultraconserved RNA genes and expand the role of non-coding RNA in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4086.

Low- and high-level expressions of heme oxygenase-1 in cultured cells under uninduced conditions

Heme oxygenase-1 (HO-1) degrades heme into biliverdin, iron, and CO. The enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. We examined the regulation of HO-1 expression in culture cells under uninduced conditions. Observations by in situ hybridization and immunostaining showed that in cultured mouse fibroblast Balb/3T3 cells not subjected to treatment, 10–15% of cells highly expressed HO-1. The similar pattern of the expression of HO-1 was observed with mouse embryo liver BNL-CL2 cells and Chinese hamster ovary cells. The marked expression of HO-1 was related to the activation of stress-activated protein kinase and to the expression of cyclooxygenase (Cox)-2. When the cells were treated with arachidonic acid, a precursor of prostaglandin, induction of HO-1 in the HO-1-expressing cells but not in the low-expressing cells occurred. This increase was abrogated by the treatment with the Cox inhibitors, indomethacin, and dexamethasone. Neither prostaglandin H 2, E 2 nor F 2a induced HO-1 expression. These results suggest that some cells respond to the cellular stress and intermediates of prostaglandin biosynthesis may act as endogenous stressors to induce HO-1.

Involvement of ABC7 in the biosynthesis of heme in erythroid cells: interaction of ABC7 with ferrochelatase

AbstractA mitochondrial half-type ATP-binding cassette (ABC) protein, ABC7, plays a role in iron homeostasis in mitochondria, and defects in human ABC7 were shown to be responsible for the inherited disease X-linked sideroblastic anemia/ataxia. We examined the role of ABC7 in the biosynthesis of heme in erythroid cells where hemoglobin is a major product of iron-containing compounds. RNA blots showed that the amount of ABC7 mRNA in dimethylsulfoxide (Me2SO)-treated mouse erythroleukemia (MEL) cells increased markedly in parallel with the induction of the mRNA expression of ferrochelatase, the last enzyme in the pathway to synthesize heme. The transfection of the antisense oligonucleotide to mouse ABC7 mRNA into Me2SO-treated MEL cells led to a decrease of heme production, as compared with sense oligonucleotide–transfected cells. ABC7 protein was shown to be colocalized with ferrochelatase in mitochondria, as assessed by immunostaining. Furthermore, in vitro and in vivo pull-down assays revealed that ABC7 protein is interacted with the carboxy-terminal region containing the iron-sulfur cluster of ferrochelatase. The transient expression of ABC7 in mouse embryo liver BNL-CL2 cells resulted in an increase in the activity and level of ferrochelatase and thioredoxin, a cytosolic protein containing iron-sulfur. These increases were also observed in MEL cells stably expressing ABC7. When ABC7 transfectants were treated with Me2SO, an increase in cellular heme concomitant with a marked induction of the expression of ferrochelatase was observed. The extent of these increases was 3-fold greater than in control cells. The results indicated that ABC7 positively regulates not only the expression of extramitochondrial thioredoxin but also that of an intramitochondrial iron-sulfur–containing protein, ferrochelatase. Then, the expression of ABC7 contributes to the production of heme during the differentiation of erythroid cells.

NITRIC OXIDE PREVENTS THE IFN-γ/LPS-INDUCED HEPATOTOXICITY IN A PROTEIN KINASE G-INDEPENDENT MANNER

Although it has been well known that the role of LPS on hepatotoxicity is mediated through TNF-α, the direct cytotoxic effect of LPS on IFN-γ-primed hepatocytes has not yet been clearly demonstrated. Here, we demonstrate that the IFN-γ-mediated death of murine embryonic liver BNL CL2 cells is potentiated by LPS (0.5 μg/ml). In addition, an exogenous NO donor, sodium nitroprusside (SNP) significantly prevents cell death induced by IFN-γ alone or IFN-γ plus LPS (IFN-γ/LPS) in a dose-dependent manner over 25 μM. SNP significantly blocked the death of BNL CL2 cells only when it was added within 12 hr after treatment of IFN-γ and IFN-γ/LPS. The preventive effect of SNP occurred in parallel with the suppression of caspase 3-like protease activation. We have also demonstrated that a relatively high concentration as well as an appropriate period of exposure to NO may be critical to maintain cell viability from the cytotoxic effect of IFN-γ and IFN-γ/LPS. Furthermore, the preventive effect of SNP on IFN-γ/LPS-induced cell death is mediated by a protein kinase G (PKG)-independent manner.

Butachlor, a suspected carcinogen, alters growth and transformation characteristics of mouse liver cells.

Butachlor is a widely used herbicide in Asia and South America. Previous investigations have indicated that it is a suspected carcinogen. To understand more about the biological effects of butachlor on cultured cells and the mechanism(s) of its carcinogenicity, we studied the alteration of the growth characteristics that was induced by butachlor in normal mouse liver cells (BNL CL2). This study demonstrates that butachlor decreases the population-doubling time of BNL CL2 cells, suggesting that it stimulates cell proliferation. To support this finding, a thymidine incorporation assay was conducted and a similar result that butachlor stimulates cell proliferation was elucidated. In addition, we show that butachlor increases the saturation density of the BNL CL2 cells. When combined with the tumor initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), butachlor transforms cells efficiently, as demonstrated by loss of contact inhibition. These findings indicate that butachlor alters the growth characteristics of BNL CL2 cells and suggest that butachlor may induce malignant transformation through stimulation of cell proliferation, alteration of cell cycle regulation, and suppression of cell density-dependent inhibition of proliferation.

ACTIVATION OF MITOGEN ACTIVATED PROTEIN KINASE IN HUMAN PLATELETS BY GENISTEIN

Genistein, a putative tyrosine kinase inhibitor, stimulated human platelet mitogen activated protein kinase (MAPK) activity in a dose- and time-dependent manner. When MAPK was maximally stimulated by phorbol 12-myristate 13-acetate (PMA), genistein still elicited the increase in MAPK activity. Staurosporine (50 nm), significantly decreased the PMA-induced MAPK activity, but had little inhibitory effect on the genistein-induced MAPK activity. Both these observations indicated a protein kinase C (PKC) independent pathway for the genistein-stimulated MAPK activity. When other tyrosine kinase inhibitors (methyl-2,5-dihydroxycinnamate, and tyrphostin) were employed, similar increases in the MAPK activity were observed. Addition of genistein to cytosolic fraction of platelets had no effect on the MAPK activity and indicated that this effect is not due to direct physical interaction between genistein and MAPK and that intact platelets are required for it. MAPK activity of platelets from rabbit and pig was also stimulated by genistein. This effect of genistein was not observed in other cell types tested (BNLCL2, HEL and U937 cells). Forskolin, which increases cyclic AMP had little effect on the basal platelet MAPK activity or the genistein activated MAPK, while it decreased by half the PMA-induced MAPK activity. The inactive analog of genistein, daidzein, which does not inhibit tyrosine kinase had little effect on MAPK. Genistein caused a decrease in basal tyrosine phosphorylation of pp60c-srcprotein as detected with anti-phosphotyrosine (anti-PTyr) Ab. Thus, inhibition of basal tyrosine kinase results in an increase in MAPK activity. This study demonstrates for the first time a novel mechanism for regulation of MAPK in platelets in which inhibition of tyrosine kinase results in activation of MAPK, independent of PKC and cAMP pathways.